Proteases are a class of enzymes, which play an important role in the processing of proteins. The body uses this mechanism to control several critical pathways or biochemical cascades, such as blood clot formation and complement activation. In neurons, specific proteases control pathways critical to neuronal communication and survival. Abnormal neuronal protease activity can lead to degenerative processes, as occurs during progressive disorders such as Alzheimer's disease and in phases of acute neuronal cell death resulting from head trauma and ischemia due to stroke. For example, these proteases can generate products that are neurotoxic, such as the amyloid beta protein (“Aβ”) which forms the senile plaques seen in Alzheimer's disease patients, or initiate degradative cascades that are involved in breaking down the neuronal cytoskeleton, leading to nerve cell death.
The term “protease” is synonymous with “peptidase”. Proteases comprise two groups of enzymes: the endopeptidases which cleave peptide bonds at points within the protein, and the exopeptidases, which remove amino acids sequentially from either N or C-terminus respectively. The term proteinase is also used as a synonym for endopeptidase. The four mechanistic classes of proteinases are: serine proteinases, cysteine proteinases, aspartic proteinases, and metallo proteinases. In addition to these four mechanistic classes, there is a section of the enzyme nomenclature which is allocated for proteases of unidentified catalytic mechanism. This indicates that the catalytic mechanism has not been identified. Thus, the possibility remains that novel types of proteases do exist.
The serine proteinases include two distinct families. The chymotrypsin family which includes the mammalian enzymes such as chymotrypsin, trypsin or elastase or kallikrein and the substilisin family which includes the bacterial enzymes such as subtilisin. The general 3D structure is different in the two families but they have the same active site geometry and then catalysis proceeds via the same mechanism. The serine proteinases exhibit different substrate specificities which are related to amino acid substitutions in the various enzyme subsites interacting with the substrate residues. Some enzymes have an extended interaction site with the substrate whereas others have a specificity restricted to the P1 substrate residue. Three residues which form the catalytic triad are essential in the catalytic process i.e His 57, Asp 102 and Ser 195 (chymotrypsinogen numbering). The first step in the catalysis is the formation of an acyl enzyme intermediate between the substrate and the essential serine. Formation of this covalent intermediate proceeds through a negatively charged tetrahedral transition state intermediate and then the peptide bond is cleaved. During the second step or deacylation, the acyl-enzyme intermediate is hydrolyzed by a water molecule to release the peptide and to restore the Ser-hydroxyl of the enzyme. The deacylation which also involves the formation of a tetrahedral transition state intermediate, proceeds through the reverse reaction pathway of acylation. A water molecule is the attacking nucleophile instead of the Ser residue. The His residue provides a general base and accept the OH group of the reactive Ser.
The cysteine proteinases includes the plant proteases such as papain, actinidin or bromelain, several mammalian lysosomal cathepsins, the cytosolic calpains (calcium-activated) as well as several parasitic proteases (e.g Trypanosoma, Schistosoma). Papain is the archetype and the best studied member of the family. Recent elucidation of the X-ray structure of the Interleukin-1-beta Converting Enzyme has revealed a novel type of fold for cysteine proteinases. Like the serine proteinases, catalysis proceeds through the formation of a covalent intermediate and involves a cysteine and a histidine residue. The essential Cys25 and His 159 (papain numbering) play the same role as Ser195 and His57 respectively. The nucleophile is a thiolate ion rather than a hydroxyl group. The thiolate ion is stabilized through the formation of an ion pair with neighboring imidazolium group of His159. The attacking nucleophile is the thiolate-imidazolium ion pair in both steps and then a water molecule is not required.
Most of aspartic proteinases belong to the pepsin family. The pepsin family includes digestive enzymes such as pepsin and chymosin as well as lysosomal cathepsins D and processing enzymes such as renin, and certain fungal proteases (penicillopepsin, rhizopuspepsin, endothiapepsin). A second family comprises viral proteinases such as the protease from the AIDS virus (HIV) also called retropepsin. Crystallographic studies have shown that these enzymes are bilobed molecules with the active site located between two homologous lobes. Each lobe contributes one aspartate residue of the catalytically active diad of aspartates. These two aspartyl residues are in close geometric proximity in the active molecule and one aspartate is ionized whereas the second one is unionized at the optimum pH range of 2-3. Retropepsins, are monomeric, i.e carry only one catalytic aspartate and then dimerization is required to form an active enzyme. In contrast to serine and cysteine proteases, catalysis by aspartic proteinases do not involve a covalent intermediate though a tetrahedral intermediate exists. The nucleophilic attack is achieved by two simultaneous proton transfer: one from a water molecule to the diad of the two carboxyl groups and a second one from the diad to the carbonyl oxygen of the substrate with the concurrent CO-NH bond cleavage. This general acid-base catalysis, which may be called a “push-pull” mechanism leads to the formation of a noncovalent neutral tetrahedral intermediate.
The metallo proteinases may be one of the older classes of proteinases and are found in bacteria, fungi as well as in higher organisms. They differ widely in their sequences and their structures but the great majority of enzymes contain a zinc atom which is catalytically active. In some cases, zinc may be replaced by another metal such as cobalt or nickel without loss of the activity. Bacterial thermolysin has been well characterized and its crystallographic structure indicates that zinc is bound by two histidines and one glutamic acid. Many enzymes contain a specific sequence which provides two histidine ligands for the zinc whereas the third ligand is either a glutamic acid (thermolysin, neprilysin, alanyl aminopeptidase) or a histidine (astacin). Other families exhibit a distinct mode of binding of the Zn atom. The catalytic mechanism leads to the formation of a noncovalent tetrahedral intermediate after the attack of a zinc-bound water molecule on the carbonyl group of the scissile bond. This intermediate is further decomposed by transfer of the glutamic acid proton to the leaving group.
Studies in a number of organisms has shown that cell death is under genetic control, and that components that regulate cell survival and death are evolutionarliy conserved, indicating that a universal cell death program exists in multicellular organisms. Apoptosis was recognized as death by an orchestrated sequence of cuts performed by enzymes which degrade macromolecular structures like DNA. A central step in carrying out cell death is the activation of members of a family of cysteine-dependent-specific proteases, now known as caspases (Nicholson and Thomberry, TIBS 22:299, 1997, herein incorporated by reference). Caspases are cysteine proteases bearing an active site with a conserved amino acid sequence and which cleave specifically following aspartate residues. These proteases were dubbed caspase as they are cysteine dependent) and caspartate cleaving protease. Caspases sufficient to carry out cell death can be expressed ubiquitously, indicating that their activation and activity must be tightly controlled in normal cells (Weil, M., et al., J Cell Biol. 133:1053, 1996). Caspase-1 (formerly called ICE, interleukin 1b-converting enzyme) was the first member of the caspase family to be identified as a protease involved in mammalian apoptosis because of its homology with CED-3. The latter gene product was known for its requirement in the execution of apoptosis in the nematode C. elegans (Yuan, et al., Cell 75:641-652, 1993). To date ten mammalian members of this family have been identified by structure and substrate specificity. There is also evidence that caspases may play roles in processes other than cell death (e.g., Song et al., Science 275:536, 1997).
Viral and cellular activators and inhibitors of protease activation or activity have been identified (e.g., Nicholson and Thomberry, supra). For caspase, many of these proteins were initially identified as regulators of apoptosis, and were subsequently tested for their ability to regulate caspase function. The present invention provides a novel fusion polypeptides and methods useful in the isolation of proteases, and in the identification of protease inhibitors.